Pharmaceutical Composition for Treating or Preventing Degenerative and Inflammatory Diseases

ABSTRACT

The present invention relates to a pharmaceutical composition useful for treating or preventing inflammatory disease and cell damage, and a method for treating or preventing inflammatory disease and cell damage. The present invention uses the 2-hydroxybenzoic acid derivative represented by the specific chemical formula or its pharmaceutically acceptable salt. The compound of the present invention is useful for treating or preventing cell damage and inflammatory disease including gastritis, gastric ulcer, pancreatitis, colitis, arthritis, diabetes, arteriosclerosis, nephritis, hepatitis, Alzheimer&#39;s disease, Parkinson&#39;s disease and Lou Gehrig&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 12/296,747 filedOct. 10, 2008, the entirety of which is incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition and amethod, which are useful for treating or preventing inflammation andcell damage occurring in inflammatory disease such as gastritis, gastriculcer, pancreatitis, colitis, arthritis, diabetes, arteriosclerosis,nephritis, hepatitis, Alzheimer's disease, Parkinson's disease and LouGehrig's disease.

BACKGROUND ART

Inflammation is reactions of blood membrane and cell against injuryfactor originated from injured cell and foreign material entered intobody. 1) Metabolites of arachidonic acid, that is, prostaglandin,leukotriene, and lipoxins, 2) platelet activation factor, 3) cytokineslike tumor necrosis factor-alpha, interleukin-1 (IL-1), etc. andchemokines like monocyte chemo-attractant protein (MCP-1), macrophageinflammatory protein-1alpha (MCP-1alpha), etc., 4) nitric oxide (NO), 5)reactive oxygen, 6) vasodilating factors such as histamine, serotonin,etc. are known to be materials intervening inflammation reaction. Mainpurpose of inflammation reaction is to remove extraneous material andinjured cell (or cell tissue), but inflammation reaction can be a causeof chronic diseases like rheumarthritis and arteriosclerosis.

In case that the degree of inflammatory damage is small, limited andtemporary, injury factors are removed and tissue comes back to normalstate according to the termination of inflammation reaction.Inflammation reaction happening where there is large damage orregeneration ability is weak is accompanied with major tissue damage,which may cause dysfunction. Chronic inflammation reaction happening fora long time may be a cause of fatal tissue damage in rheumarthritis,arteriosclerosis, tuberculosis, chronic pulmonary disease, etc.(Pathological Basis of Disease, pp 47-86, 7th edition).

In addition, there are many literatures disclosing that inflammationplays a critical role in pathophysiology of degenerative brain disease.Microglia present in brain is activated at site where neuronal damagehappens in Alzheimer's disease, Parkinson's disease and Lou Gehrig'sdisease (Liu B and Hong J S. J Pharmacol Exp Ther. 2003; 304(1):1-7; OrrC F et al., Prog Neurobiol. 2002; 68(5):325-40; and Henkel J S et al.,Ann Neurol. 2004; 55(2):221-35). Activated microglia producesprostaglandins, cytokines, chemokines, reactive oxygen species, NO,etc., which begin inflammation reaction in brain (Minghetti L. Curr OpinNeurol. 2005; 18(3):315-21; Gao H M, Trends Pharmacol Sci. 2003;24(8):395-401; Weydt P and Moller T. Neuroreport. 2005; 16(6):527-31; J.J. M. Hoozemans Int. J. Devl Neuroscience. 2006; 24:157-165). Therefore,administration of drug inhibiting inflammation is reported to suppressproduction of beta-amyloid and plague in animal model of Alzheimer'sdisease (Townsend K P and Pratico D. FASEB J. 2005; 18:315-21), protectdopaminergic neuronal cell in animal model of Parkinson's disease(Ferger B et al., Naunyn Schmiedebergs Arch Pharmacol. 1999;360(3):256-61; Teismann P, Ferger B. Synapse. 2001; 39(2):167-74),prevent death of spinal motoneuron and reduce activity of glia in LouGehrig's disease model (Kiaei M et al., J Neurochem. 2005;93(2):403-11). Actually, an attack rate of Alzheimer's disease is highin patient having brain inflammation reaction caused by contusion, etc.(Breitner J C. Neurology. 1994; 44(2):227-32), and an attack rate ofAlzheimer's disease is low in rheumarthritis patient taking nonsteroidalanti-inflammatory drugs (NSAIDS) for a long time (McGeer P L et al.,Lancet. 1990; 335(8696):1037). In addition, it is reported thatadministration of NSAID not only prevents Alzheimer's disease, but alsodelays progress of damage of cognitive function (Rich et al., Neurology.1995). These results suggest that a drug inhibiting inflammationreaction may be used for preventing and treating degenerative braindisease.

NSAIDs, drugs suppressing activity of cyclooxygenase taking part inproduction of prostaglandin, have been developed and widely used toalleviate symptoms, including pain, of inflammatory diseases, but thereare side effects to block the use of the NSAIDs. Specifically,gastrointestinal disorders such as dyspepsia, gastritis, ulcer, bleedingand perforation are side effects often happening after administration ofNSAIDs. Actually, because of adverse effects of NSAIDs, 107,000 ofpeople are reported to be hospitalized and 16,500 of people are reportedto be dead in USA only. Celecoxib and Rofecoxib, selective COX-2(cyclooxygenase-2) enzyme inhibitors, having low side effects ongastrointestinal damage have been developed and used for treatingarthritis and pain. However, U.S. FDA reported that long-termadministration of celecoxib, rofecoxib and valdecoxib might cause heartdisease, and prohibited the use of these as drug for treating arthritis.Furthermore, clinical trials to evaluate therapeutic efficacy ofcelecoxib and rofecoxib in dementia were discontinued because of theirsafety.

In addition, reactive oxygen species produced by neutrophil, macrophage,monocyte, etc. in inflammatory disease is known to be a major reasoncausing tissue damage by mediating inflammatory reaction. In actually,administration of drugs removing reactive oxygen are reported to beeffective in treating gastric damage happing in inflammatory diseases(Matthews G M et al., Helicobacter. 2005; 10(4):298-306), pancreaticdamage (Shi C et al., Pancreatology. 2005; 5(4-5):492-500),atherosclerosis (Tardif J C. Curr Atheroscler Rep. 2005; 7(1):71-7),colon damage (Oz H S et al., J Nutr Biochem. 2005; 16(5):297-304), jointdamage (Henrotin Y E et al., Osteoarthritis Cartilage. 2003;11(10):747-55), renal damage (Tian N et al., Hypertension. 2005;45(5):934-9), river damage (Loguercio C et al., Free Radic Biol Med.2003; 34(1):1-10) and cardiovascular damage (Haidara M A et al., CurrVasc Pharmacol. 2006; 4(3):215-27). Furthermore, administration ofNSAIDs causes production of reactive oxygen, and induces damage to thegastric mucous membrane. This gastric damage is reported to be lessenedby administration of anti-oxidant material (Graziani G et al., Gut.2005; 54(2):193-200).

Inflammation plays an important role in pathophysiology of digestivedisease, respiratory disease or neuronal system disease, but the use ofcurrently available drugs is limited because of the side effects of thedrugs. Aspirin (acetylsalicylic acid), an anti-inflammatory drug, isknown to suppress the action of NF-kB and c-Jun N-terminal kinase (Ko HW et al., J Neurochem. 1998; 71(4):1390-5), and sulfasalazine is knownto protect cell by its anti-oxidant activity (Ryu B R et al., JPharmacol Exp Ther. 2003; 305(1):48-56). However, there is a drawbackthat aspirin and sulfasalazine show their cell-protecting effects inhigh concentration only.

DISCLOSURE Technical Problem

Accordingly, the object of the present invention is to provide apharmaceutical composition useful for treating or preventinginflammatory disease and having no side effects such as gastric damageand cardiovascular disorder, a treating or preventing use of thecomposition against inflammatory disease, and a method for treating orpreventing inflammatory disease, comprising administering thecomposition

TECHNICAL SOLUTION

To achieve the object, the present invention provides a pharmaceuticalcomposition for treating or preventing inflammatory disease, comprising2-hydroxybenzoic acid derivative represented by the below chemicalformula 1 or its pharmaceutically acceptable salt as effective agent:

wherein,

n is an integer from 2 to 5;

R₁ is hydrogen or alkyl;

R₂ is hydrogen, alkyl or alkanoyl;

R₃ is hydrogen or acetoxy; and

X is independently hydrogen, nitro, halogen, alkyl, haloalkyl, alkoxy orhaloalkoxy.

More preferably, the present invention provides the pharmaceuticalcomposition, wherein the inflammatory disease is any one selected fromthe group consisting of gastritis, colitis, arthritis, diabeticinflammation, arteriosclerosis, nephritis, hepatitis, Alzheimer'sdementia, Parkinson's disease and Lou Gehrig's disease.

The present invention also provides a method for treating or preventinginflammatory disease, comprising administering to a patient or animalsuffering from inflammatory disease a therapeutically effective amountof 2-hydroxybenzoic acid derivative represented by the chemical formula1 or its pharmaceutically acceptable salt.

The present inventors have prepared and evaluated a lot of compounds,and succeeded in inventing the fact that the 2-hydroxybenzoic acidderivative or its pharmaceutically acceptable salt is much useful fortreating or preventing inflammatory disease as well as safe.

Hereinafter, the pharmaceutical composition for treating or preventinginflammatory disease and the method for treating or preventinginflammatory disease will be described in more detail.

The present invention provides a new use, that is, a use for treating orpreventing inflammatory disease, of the 2-hydroxybenzoic acid derivativerepresented by the chemical formula 1 or its pharmaceutically acceptablesalt, and also provides a method for treating or preventing inflammatorydisease, using the 2-hydroxybenzoic acid derivative or itspharmaceutically acceptable salt.

wherein,

n is an integer from 2 to 5;

R₁ is hydrogen or alkyl;

R₂ is hydrogen, alkyl or alkanoyl;

R₃ is hydrogen or acetoxy; and

X is independently hydrogen, nitro, halogen, alkyl, haloalkyl, alkoxy orhaloalkoxy.

Preferably, in the chemical formula 1, alkyl (including ‘alkyl’ ofhaloalkyl) is C₁-C₅ alkyl, and more preferably C₁₋C₃ alkyl. Morespecifically, preferable alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl. Alkoxy(including ‘alkoxy’ of haloalkoxy), preferably, is C₁-C₅ alkoxy, andmore preferably C₁-C₃ alkoxy. More specifically, preferable alkoxyincludes, but is not limited to, methoxy, ethoxy, and propanoxy. Halogenincludes, but is not limited to, fluoride, chloride, bromide, andiodide. Preferably, alkanoyl is C₂-C₁₀ alkanoyl, and more preferablyC₃-C₅ alkanoyl. More specifically, preferable alkanoyl includes, but isnot limited to, ethanoyl, propanoyl, and cyclohexanecarbonyl.

Preferable examples of the 2-hydroxybenzoic acid derivative include, butare not limited to, 2-hydroxy-5-phenethylamino-benzoic acid (compound1), 2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 2),2-hydroxy-5-[2-(3-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 3),5-[2-(3,5-bis-trifluoromethyl-phenyl)-ethylamino]-2-hydroxy-benzoic acid(compound 4), 2-hydroxy-5-[2-(2-nitro-phenyl)-ethylamino]-benzoic acid(compound 5), 5-[2-(4-chloro-phenyl)-ethylamino]-2-hydroxy-benzoic acid(compound 6), 5-[2-(3,4-difluoro-phenyl)-ethylamino]-2-hydroxy-benzoicacid (compound 7),5-[2-(3,4-dichloro-phenyl)-ethylamino]-2-hydroxy-benzoic acid (compound8),5-[2-(4-fluoro-2-trifluoromethyl-phenyl)-ethylamino]-2-hydroxy-benzoicacid (compound 9),5-[2-(2-fluoro-4-trifluoromethyl-phenyl)-ethylamino]-2-hydroxy-benzoicacid (compound 10),2-hydroxy-5-[2-(4-methoxy-phenyl)-ethylamino]-benzoic acid (compound11), 2-hydroxy-5-(2-o-tolyl-ethylamino)-benzoic acid (compound 12),2-hydroxy-5-(3-phenyl-propylamino)-benzoic acid (compound 13),2-hydroxy-5-[3-(4-trifluoromethyl-phenyl)-propylamino]-benzoic acid(compound 14), 5-[3-(4-fluoro-phenyl)-propylamino]-2-hydroxy-benzoicacid (compound 15),5-[3-(3,4-dichloro-phenyl)-propylamino]-2-hydroxy-benzoic acid (compound16), 2-hydroxy-5-(3-p-tolyl-propylamino)-benzoic acid (compound 17),2-acetoxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 18), and 5-[2-(2-chloro-phenyl)-ethylamino]-2-hydroxy-benzoicacid (compound 19).

Among the preferable compounds above,2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 2) and2-acetoxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 18) are more preferable as therapeutic agent for treatinginflammatory disease than other 2-hydroxybenzoic acid derivatives.

Particularly, the compound 2 is more preferable than other2-hydroxybenzoic acid derivatives when used for treating cell damage andinflammation of degenerative brain diseases. Compound 2 showed superioranti-inflammatory effect and superior suppressing effect on productionof beta-amyloid compared to some compounds having similar chemicalstructure. Even if there are compounds showing better effect thancompound 2 when considering one test only like suppressing effect onproduction of NO, but the compounds are not better as therapeutic agentfor treating degenerative brain diseases than compound 2 because thecompounds showed relatively worse effects in the other tests (forexample, some compounds showed better suppressing effects on productionof NO than compound 2, but the compounds showed very weak suppressingeffect on production of beta-amyloid, which is very important factor intreating degenerative brain disease). In addition, some compounds showedgood therapeutic efficacy in all efficacy tests, but they showed badsafety results compared to compound 2 like the following toxicity test.

Similarly, among the preferable compounds, compound 18 is morepreferable in treating inflammatory disease than other 2-hydroxybenzoicacid derivatives. Compound 18 showed very much superior effect inanti-inflammatory efficacy tests compared to compounds having similarstructure, and is less preferable than compound 2 in the followingtoxicity test.

The 2-hydroxybenzoic acid derivative of the present invention can beprepared by, but is not limited to, the below reaction schemes. The2-hydroxybenzoic acid derivative of the present invention can beprepared by the conventional synthesis method well known in the art towhich the present pertains.

In addition,2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 2), one preferable example of the present invention, can beprepared by, but is not limited to, the following reaction scheme 3.

The term “pharmaceutically acceptable salt” of the present inventionmeans salts produced by non-toxic or little toxic base. In case that thecompound of the present invention is acidic, base addition salts of thecompound of the present invention can be made by reacting the free baseof the compound with enough amount of desirable base and adequate inertsolvent. Pharmaceutically acceptable base addition salt includes, but isnot limited to, sodium, potassium, calcium, ammonium, magnesium or saltmade by organic amino. In case that the compound of the presentinvention is basic, acid addition salts of the compound of the compoundcan be made by reacting the free base of the compound with enough amountof desirable acid and adequate inert solvent. Pharmaceuticallyacceptable acid addition salt includes, but is not limited to, propionicacid, isobutylic acid, oxalic acid, malic acid, malonic acid, benzoicacid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalicacid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaricacid, methanesulfonic acid, hydrochloric acid, bromic acid, nitric acid,carbonic acid, monohydrogencarbonic acid, phosphoric acid,monohydrogen-phosphoric acid, dihydrogen-phosphoric acid, sulfuric acid,monohydrogen-sulfuric acid, hydrogen iodide, and phosphorous acid. Inaddition, the pharmaceutically acceptable salt of the present inventionincludes, but is not limited to, a salt of amino acid like arginate andan analog of organic acid like glucuronic or galactunoric.

For example, a pharmaceutically acceptable salt of2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 2), one preferable example of the present invention, can beprepared by the below reaction scheme 4. However, the following reactionmethods are offered by way of illustration and are not intended to limitthe scope of the invention.

In the scheme, M is a pharmaceutically acceptable metal or basic organiccompound such as diethylamine, lithium, sodium and potassium.

In more detail, diethylamine salt can be prepared by dissolving acompound in alcohol, adding dropwise diethylamine, stirring the mixture,distilling in vacuo, and crystallizing the residue by adding ether.Alkali metal salt can be made by preparing desirable salt with inorganicreagent like lithium hydroxide, sodium hydroxide, potassium hydroxide insolvent like alcohol, acetone, acetonitrile and then freeze-drying. Inaddition, according to the similar method, lithium salt can be made withlithium acetate, sodium salt can be made with sodium 2-ethylhexanoate orsodium acetate, and potassium salt can be made with potassium acetate.

Some of the compounds of the present invention may be hydrated form, andmay exist as solvated or unsolvated form. A part of compounds accordingto the present invention exist as crystal form or amorphous form, andany physical form is included in the scope of the present invention. Inaddition, some compounds of the present invention may contain one ormore asymmetric carbon atoms or double bond, and therefore exists in twoor more stereoisomeric forms like racemate, enantiomer, diastereomer,geometric isomer, etc. The present invention includes these individualstereoisomers of the compounds of the present invention.

The present invention also provides a pharmaceutical compositioncomprising the 2-hydroxybenzoic acid derivative represented by the abovechemical formula 1 or its pharmaceutically acceptable salt; andpharmaceutically acceptable excipient or additive. The compound or itspharmaceutically acceptable salt of the present invention may beadministered alone or with any convenient carrier, diluent, etc. and aformulation for administration may be single-dose unit or multiple-doseunit.

The pharmaceutical composition of the present invention may beformulated in a solid or liquid form. The solid formulation includes,but is not limited to, a powder, a granule, a tablet, a capsule, asuppository, etc. Also, the solid formulation may further include, butis not limited to, a diluent, a flavoring agent, a binder, apreservative, a disintegrating agent, a lubricant, a filler, etc. Theliquid formulation includes, but is not limited to, a solution such aswater solution and propylene glycol solution, a suspension, an emulsion,etc., and may be prepared by adding suitable additives such as acoloring agent, a flavoring agent, a stabilizer, a thickener, etc.

For example, a powder can be made by simply mixing the 2-hydroxybenzoicacid derivative of the present invention and pharmaceutically acceptableexcipients like lactose, starch, microcrystalline cellulose. A granulecan be prepared as follows: mixing the compound or its pharmaceuticallyacceptable salt, a pharmaceutically acceptable diluent and apharmaceutically acceptable binder such as polyvinylpyrrolidone,hydroxypropylcellulose, etc; and wet-granulating with adequate solventlike water, ethanol, isopropanol, etc, or direct-compressing withcompressing power. In addition, a tablet can be made by mixing thegranule with a pharmaceutically acceptable lubricant such as magnesiumstearate, and tabletting the mixture.

The pharmaceutical composition of the present invention may beadministered in forms of, but not limited to, oral formulation,injectable formulation (for example, intramuscular, intraperitoneal,intravenous, infusion, subcutaneous, implant), inhalable, intranasal,vaginal, rectal, sublingual, transdermal, topical, etc. depending on thedisorders to be treated and the patient's conditions. The composition ofthe present invention may be formulated in a suitable dosage unitcomprising a pharmaceutically acceptable and non-toxic carrier, additiveand/or vehicle, which all are generally used in the art, depending onthe routes to be administered. Depot type of formulation being able tocontinuously release drug for desirable time also is included in thescope of the present invention.

The present invention also provides a use of the 2-hydroxybenzoic acidderivative or its pharmaceutically acceptable salt for treating and/orpreventing inflammatory disease. That is, the present invention providesa pharmaceutical composition for treating or preventing inflammatorydisease, comprising the 2-hydroxybenzoic acid derivative represented bythe above chemical formula 1 or its pharmaceutically acceptable salt.More specifically, the 2-hydroxybenzoic acid derivative or itspharmaceutically acceptable salt can be used for treating or preventinginflammatory disease such as gastritis, gastric ulcer, pancreatitis,colitis, arthritis, diabetes, arteriosclerosis, nephritis and hepatitis,and cell damage and inflammation occurring in degenerative brain diseasesuch as Alzheimer's dementia, Parkinson's disease and Lou Gehrig'sdisease. However, the use of the 2-hydroxybenzoic acid derivative or itspharmaceutically acceptable salt according to the present invention isnot limited to the above concrete disease names.

For treating inflammatory disease, the compound of the present inventionmay be administered daily at a dose of approximately 0.01 mg/kg toapproximately 100 g/kg, preferably approximately 0.1 mg/kg toapproximately 10 g/kg. However, the dosage may be varied according tothe patient's conditions (age, sex, body weight, etc.), the severity ofpatients in need thereof, the used effective components, diets, etc. Thecompound of the present invention may be administered once a day orseveral times a day in divided doses, if necessary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that compound 2 did not cause damage to the gastric mucousmembrane, which means that compound 2 is safe. Test samples were orallyadministered at different doses to evaluate the degree of the gastricmucous membrane damage. Aspirin was used as control.

FIG. 2 is results showing degrees of the gastric mucous membrane damage.Test samples were orally administered at indicated doses. Compound 18did not cause gastric mucous membrane damage at high dose.Indomethacine, ibuprofen and celecoxib were used as control.

FIG. 3 is results showing cell-protecting effect of compound 2 ongastric mucous membrane damage induced by Helicobacter. Helicobacter(43504, 5×10⁸ cfu/ml) was administered to cultured AGS (human gastriccancer) cell, alone or with sample having indicated concentration. MTTanalysis method was used to evaluate the survival rate of gastric mucousmembrane cell 24 hours after administration.

FIG. 4 is results showing cell-protecting effect of compound 2 ongastric mucous membrane damage induced by sulindac, a NSAID. 100 uM ofsulindac was administered to cultured AGS cell, alone or with samplehaving indicated concentration. MTT analysis method was used to evaluatethe survival rate of gastric mucous membrane cell 16 hours afteradministration.

FIG. 5 is results showing cell-protecting effect of compound 2 ongastric mucous membrane damage induced by H₂O₂, oxidative stress.Cultured AGS cell was treated with 100 uM of H₂O₂ for 8 hours, alone orwith sample having indicated concentration, and was washed with culturemedium. MTT analysis method was used to evaluate the survival rate ofgastric mucous membrane cell 24 hours later.

FIG. 6 is results showing cell-protecting effect of compound 2 ongastric mucous membrane damage induced by alcohol (ethanol). CulturedAGS cell was treated with 25 mM of alcohol for 24 hours, alone or withsample having indicated concentration. Then, MTT analysis method wasused to evaluate the survival rate of gastric mucous membrane cell.

FIG. 7 is a picture showing the expression degree of TNF-α mRNA.Cultured AGS cell was treated with 25 mM of alcohol for 24 hours, aloneor with compound 2 having indicated concentration. Then, mRNA wasextracted and evaluated by RT-PCR method.

FIG. 8 is results showing protecting effect of compound 2 ongastrointestinal bleeding caused by alcohol/hydrochloric acid(EtOH/HCl). A is a picture of normal rat stomach. B is a picture of ratstomach, wherein gastrointestinal bleeding was caused by oraladministration of 60% EtOH/150 mM HCl after 200˜250 g of rat was fastedfor 24 hours. C is a picture of rat stomach, wherein rat was pre-treatedwith compound 230 minutes before administration of alcohol/HCl, andstomach was taken out 90 minutes after gastrointestinal bleeding. D is agraph quantifying damaged area to evaluate the degree of stomach injury.

FIG. 9 is results showing protecting effect of compound 2 on alcoholicgastritis. A is a picture of normal rat stomach. B is a picture of ratstomach, wherein gastrointestinal bleeding was caused by oraladministration of ethanol (4 ml/kg) after 250 g of rat was fasted for 24hours. C is a picture of rat stomach, wherein rat was pre-treated withcompound 21 hour before induction of gastrointestinal bleeding, andstomach was taken out 90 minutes after gastrointestinal bleeding. D is agraph quantifying the degree of stomach injury.

FIG. 10 is results showing protecting effect of compound 2 on gastritisinduced by NSAID. 250 g of rat was fasted for 24 hours, and indomethacinwas orally administered to cause gastrointestinal bleeding (A and C).Compound 2 was administered 1 hour before administration of NSAID (B andD), and stomach was taken out 6 hours (A and B) or 12 hours (C and D)after gastrointestinal bleeding.

FIG. 11 is results showing efficacy of compound 2 through thermalhyperalgesia induced by carrageenan, one of arthritis animal models. 2%carrageenan was intradermally injected into hindpaw of rat to inducethermal hyperalgesia. Compound 2 was orally administered, and ibuprofenwas used as comparative example.

FIG. 12 is results showing efficacy of compound 2 through the levels ofTNF-alpha induced by zymosan, one of arthritis animal models. 1% zymosanwas administered with air pouch, and compound 2 was once orallyadministered according to indicated dose 1 hour before administration ofzymosan. The level of TNF-alpha in an exudation of air pouch wasevaluated by ELISA method 4 hours later.

FIG. 13 is graphs showing how much compound 2 decreases the level ofIL-1alpha induced by zymosan, one of arthritis animal models. 1% zymosanwas administered with air pouch, and compound 2 was once orallyadministered according to indicated dose 1 hour before administration ofzymosan. The level of IL-1alpha in an exudation of air pouch wasevaluated by ELISA method 4 hours later.

FIG. 14 is results showing efficacy through arthritis animal modelinduced by collagen. FIG. 14 shows edema of foot by collagen.

Sham: normal mouse

Collagen: mouse administered collagen

Compound 18: mouse intraperitoneally injected with 25 mg/kg of compound18

FIG. 15 is results showing efficacy through arthritis animal modelinduced by collagen. 25 mg/kg of compound 2, 3 or 18, or methotrexate(control, MTX, 1 mg/kg/week) was intraperitoneally injected afteradministration of collagen. Then, mice were observed with the naked eyefor 4 weeks, and results were evaluated by arthritis index.

FIG. 16 is results showing efficacy of compounds 6 and 11 througharthritis animal model induced by collagen. Collagen was intradermallyinjected two times with 2 week-interval. One week later, compound 6,compound 11 and methotrexate (control) were intraperitoneally injected.Then, the animals were observed everyday for 2˜3 weeks, and results wasevaluated by arthritis index.

FIG. 17 is results showing efficacy through 5% dextran sulfate sodium(DSS) model, inflammatory intestinal disease animal model. Aftertreatment of DSS, 10, 25 or 50 mg/kg of compound 18 suspended in 10%vehicle was orally administered. Sham means colon of normal mouse, DSSmeans colon mouse treated with 5% DSS. The others are pictures of colonof mice administered with compound 18 at indicated dose.

FIGS. 18 and 19 are graphs quantifying results of FIG. 17. FIG. 18 islength of colon, and FIG. 19 is width of colon.

FIG. 20 is an efficacy test result of compound 18 using DSS. Aftertreatment of DSS, compound 18 (25 mg/kg) suspended in 10% vehicle wasorally administered. After that, bloody excrement by colon injury,diarrhea and the degree of dirtiness were evaluated as feces score.

FIG. 21 is efficacy test results using acute pancreatitis model inducedby cerulein. 50 ug/kg of cerulein was administered. Two hours later,edema was induced by administration of LPS. A-C are picture results, andD is graphs showing the weight of pancreas.

A: normal wistar rat

B: wistar rat administered cerulein

C: wistar rat administered compound 18 (25 mg/kg)

FIG. 22 is efficacy test results using acute pancreatitis model inducedby cerulein. 50 ug/kg of cerulein was administered. Two hours later,edema was induced by administration of LPS. 25 mg/kg of compound 18 wasadministered, and the changes of amylase and lipase, pancreaticdigestive enzymes, were quantified.

FIGS. 23 and 24 are efficacy test results using pancreatitis modelinduced by cerulein. The effects of the 2-hydroxybenzoic acid derivativeof the present invention on TNF-α, IL-1β and PGE₂, inflammatory markers,were evaluated.

FIG. 25 are graphs showing the levels of TNF-α, IL-1β or IL-6 in brainof 10.5 month-old APP/PS1 dementia mouse (Tg+). The levels wereevaluated by ELISA method. Compound 2 (25 mg/kg/day) or ibuprofen (62.5mg/kg/day) mixed with chow was fed for 7 months from 3 month-old.

FIG. 26 is pictures (low and high magnification) showing protectingeffect of compound 2 on brain-blood vessel barrier.

A: normal mouse

B/F: APP/PS1 dementia mouse

C/G: mouse administered compound 2 (25 mg/kg/day)

D/H: mouse administered ibuprofen (62.5 mg/kg/day)

FIG. 27 is pictures of amyloid plaque produced in brain of 10.5month-old normal mouse (A), 10.5 month-old APP/PS1 dementia mouse (B,Tg+), APP/PS1 dementia mouse fed with chow containing compound 2 (25mg/kg/d, C) or ibuprofen (62.5 mg/kg/d, D), for 7 months from 3.5month-old. Thioflavin-S pigment was used for staining. E is graphsquantifying results of A-D.

FIG. 28 is results of Elevated plus maze test performed with 10.5month-old normal mouse, 10.5 month-old APP/PS1 dementia mouse, andAPP/PS1 dementia mouse having administration of chow with compound 2 (25mg/kg/d) for 7 months from 3.5 month-old. The time spent in the open armwas recorded as efficacy result.

FIG. 29 is pictures showing the activity of microglia, a marker ofinflammation, in G93A mouse. The results were immunostained with TOMATOLectin.

A: normal mouse

B: G93A mouse

C: G93A mouse administered 5 mg/kg/day of compound 2

FIG. 30 is results showing the levels of mRNA of inflammatory cytokines(TNF-α and IL-1β), markers of inflammation, in G93A mouse. RT-PCR(Reverse Transcription-Polymerase Chain Reaction) was used.

FIG. 31 is pictures showing suppressing effect of compound 2 oninflammation in Parkinson's disease animal model. The results wereimmunostained with CD11b.

A: mouse administered MPTP

B: mouse administered with 50 mg/kg/d of compound 2

FIG. 32 is results of single dose toxicity testing of some2-hydroxybenzoic acid derivatives according to the present invention.

MODES FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in considerable detailto help those skilled in the art understand the present invention.However, the following examples are offered by way of illustration andare not intended to limit the scope of the invention. It is apparentthat various changes may be made without departing from the spirit andscope of the invention or sacrificing all of its material advantages.

Synthesis Example 1 Preparation of2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 2)

5-aminosalicylic acid (0.51 g, 3.90 mmole) was added toN,N-dimethylformamide (20.0 ml) at room temperature under nitrogenatmosphere, and the reaction mixture was stirred. Triethylamine (0.50ml) and tetrabutylammonium iodide (10.1 mg) were added, and stirred for30 minutes. Then, 1-(2-(bromoethyl)-4-trifluoromethyl)benzene was added,and stirred at room temperature for 3 hours. Ice was added to quench thereaction. Produced crystal was filtered, again stirred with acetone andhexane, and re-filtered. Filtered solid was dissolved in ethylacetate,washed with 0.5 N hydrochloric acid and brine, dried over magnesiumsulfate anhydrous, and distilled in vacuo to give 0.54 g (21% yield) of2-hydroxy-5-(4-(trifluoromethyl)phenylethylamino)benzoic acid.

¹H NMR (DMSO-d₆): 7.62 (d, 2H), 7.48 (d, 2H), 6.98 (d, 1H) 6.88 (q, 1H),6.76 (d, 1H), 3.24 (t, 2H), 2.91 (t, 2H)<

Synthesis Example 2 Preparation of2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acidpotassium salt

2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid (10g, 30.7 mmole) produced in the synthesis example 1 was added toanhydrous ethanol (100 ml), and warmed up to 50° C. to completelydissolve. Then, the solution was cooled to 0° C. pH was adjusted to6.8˜7.0 with the solution of 85% potassium hydroxide (2.03 g, 30.7mmole) and anhydrous ethanol (20 ml), and stirred at room temperaturefor 2 hours. Precipitated crystal was filtered and dried to give 10.4 g(93% yield) of 2-hydroxy-5-(4-(trifluoromethyl)phenylethylamino)benzoicacid potassium salt.

¹H NMR (DMSO-d₆): 7.62 (d, 2H), 7.48 (d, 2H), 6.98 (d, 1H), 6.90 (q,1H), 6.84 (d, 1H), 3.24 (t, 2H), 2.91 (t, 2H)<

Synthesis Example 3 Preparation of2-hydroxy-5-[2-(4-nitrophenyl)ethylamino]-benzoic acid

According to the similar procedure to Synthesis Example 1, by using5-aminosalicylic acid (500 mg, 3.26 mmole) and 4-nitrophenylethylbromide (900 mg, 3.92 mmole), 890 mg (50% yield) of2-hydroxy-5-[2-(4-nitrophenyl)ethylamino]-benzoic acid was obtained as apale yellow solid. Melting point 234-236° C.

Elemental analysis of C₁₅H₁₄N₂O₅

TABLE 1 % C % H % N Calculated 59.60 4.67 9.27 Found 59.77 4.79 9.24

Synthesis Example 4 Preparation of 2-hydroxy-5-[3-(4-nitrophenyl)-n-propylamino]-benzoic acid

According to the similar procedure to Synthesis Example 1, by using5-aminosalicylic acid (500 mg, 3.26 mmole) and 3-(4-nitrophenyl)propylbromide (950 mg, 3.92 mmole), 520 mg (50% yield) of2-hydroxy-5-[3-(4-nitrophenyl)-n-propylamino]-benzoic acid was obtainedas a pale yellow solid. Melting point 229-231° C.

Elemental analysis of C₁₅H₁₆N₂O₅

TABLE 2 % C % H % N Calculated 60.75 5.10 8.86 Found 60.77 5.07 8.89

Synthesis Example 5

According to the similar procedure to Synthesis Example 1, the othercompounds were prepared. Analysis results were shown in table 3 below.

TABLE 3 # ¹H-NMR (δ) compound 3 7.54 (m, 4H), 7.20 (s, 1H), 6.59 (d,1H), 6.47 (d, 1H), 3.19 (t, 2H), 2.90 (t, 2H) compound 4 7.97 (s, 2H),7.88 (s, 1H), 7.28 (s, 1H), 7.15 (t, 1H), 6.82 (d, 1H), 3.42 (t, 2H),3.09 (t, 2H) compound 5 7.91 (d, 1H), 7.58 (t, 1H), 7.51 (d, 1H), 7.43(t, 1H), 7.10 (d, 1H), 6.64 (q, 1H), 6.51 (d, 1H), 3.21 (t, 2H), 3.04(t, 2H) compound 6 7.31 (d, 2H), 7.26 (d, 2H), 7.05 (s, 1H), 6.58 (d,1H), 6.49 (d, 1H), 3.18 (t, 2H), 2.81 (t, 2H) compound 7 7.31 (m, 2H),7.11 (s, 1H), 6.94 (d, 1H), 6.87 (d, 1H), 6.74 (d, 1H), 3.17 (t, 2H),2.87 (t, 2H) compound 8 7.51 (q, 2H), 7.19 (d, 1H), 6.97 (t, 1H), 6.75(t, 1H), 6.44 (d, 1H), 3.22 (t, 2H), 2.91 (t, 2H) compound 9 7.94 (s,1H), 7.64 (q, 2H), 7.45 (q, 2H), 7.10 (d, 1H), 3.48 (t, 2H), 3.21 (t,2H) compound 7.83 (t, 1H), 7.64 (d, 1H), 7.35 (t, 1H), 6.96 (q, 1H), 106.52 (t, 1H), 3.20 (t, 2H), 3.11 (t, 2H) compound 7.32 (d, 1H), 7.14 (d,2H), 6.82 (d, 2H), 6.78 (q, 1H), 11 6.64 (d, 1H), 3.79 (s, 3H), 3.24 (t,2H), 2.80 (t, 2H) compound 7.21-7.11 (m, 5H), 6.93 (d, 1H), 6.78 (d, 1H)3.17 (t, 2H), 12 2.82 (t, 2H) compound 7.27-7.11 (m, 5H), 6.95 (d, 1H),6.81 (q, 1H), 6.70 (m, 13 1H), 2.97 (t, 2H), 2.64 (t, 2H), 1.82 (m, 2H)compound 7.61 (d, 2H), 7.43 (d, 2H), 6.97 (s, 1H), 6.85 (t, 1H), 14 6.78(d, 1H), 3.04 (t, 2H), 2.78 (t, 2H), 1.87 (m, 2H) compound 7.17 (t, 3H),7.02 (t, 2H), 6.94 (d, 1H), 6.78 (d, 1H), 15 2.99 (t, 2H), 2.63 (t, 2H),1.82 (m, 2H) compound 7.45 (q, 2H), 7.15 (d, 1H), 7.05 (d, 1H), 6.92 (q,1H), 16 6.77 (d 1H), 2.96 (t, 2H), 2.66 (t, 2H), 1.82 (m, 2H) compound7.32 (s, 1H), 7.18 (d, 1H), 7.06 (s, 4H), 6.84 (d, 1H), 17 3.01 (t, 2H),2.61 (t, 2H), 2.20 (s, 3H), 1.83 (m, 2H)

Synthesis Example 6 Preparation of2-acetoxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid(compound 18)

Compound 18 was prepared by the reaction scheme below.

Compound a: To a solution of compound 2 (50.0 g, 154 mmol) in 200 mL ofH₂O:1,4-dioxane (1:1) at room temperature were added Na₂CO₃ (32.6 g, 307mmol), di-tert-butyl dicarbonate (40.3 g, 185 mmol). After 8 hoursstirring, additional di-tert-butyl dicarbonate (16.8 g, 76.9 mmol) wasadded. After additional 8 hours stirring at room temperature, thereaction mixture was neutralized (pH ˜6) with aqueous 2 N HCl. Theresulting mixture was extracted with ethyl acetate (2×200 mL). Thecombined organic phase was washed with brine (100 mL), dried over MgSO₄and filtered. Concentration gave compound a as yellow foam. Crude a wasused without further purification.

¹H NMR (400 MHz, CDCl₃) δ 10.52 (1H, br S), 7.53 (d, 2H, J=8 Hz), 7.53(br, 1H), 7.28 (d, 2H, J=8 Hz), 7.26 (br, 1H), 6.94 (d, 1H, J=8.8 Hz),3.85 (t, 2H, J=7.2 Hz), 2.94 (t, 2H, J=7.2 Hz), 1.43 (br s, 9H).

Compound b: To a solution of compound a obtained above indichloromethane (200 mL) were added N,N-diisopropylethylamine (80.3 mL,461 mmol) and acetyl chloride (21.9 mL, 307 mmol) at 0° C. The reactionmixture was warmed to room temperature and stirred for 5 hours. Aqueous1 N HCl (100 mL) was added to the reaction mixture. The layers wereseparated and the aqueous layer was extracted with dichloromethane (100mL). The combined organic layer was washed with brine (100 mL), driedover MgSO₄, filtered and concentrated. Recrystallization of the crudeproduct in diethyl ether gave 22 (51.5 g, 71.7% for 2 steps) as a whitesolid.

¹H NMR (400 MHz, CDCl₃) δ 7.82 (br s, 1H), 7.54 (d, 2H, J=8 Hz),7.38-7.24 (m, 1H), 7.28 (d, 1H, J=8 Hz), 7.08 (d, 1H, J=7.6 Hz), 3.91(t, 2H, J=7.2 Hz), 2.98 (t, 2H, J=7.2 Hz), 2.34 (s, 3H), 1.42 (br s,9H).

Compound 18: A solution of compound b (51.5 g, 110 mmol) indichloromethane (200 mL) at 0° C. was treated with 4 N HCl in1,4-dioxane (200 mL). The reaction mixture was warmed to roomtemperature. After 5 hours stirring, the suspension was concentrated.The residue was triturated in diethyl ether (500 mL). Filtration andwashing with dichloromethane (500 mL) and with diethyl ether (500 mL)gave compound 18 (46.0 g, 82.5 mmol, 92.3%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 7.66 (d, 2H, J=7.6 Hz), 7.52 (d, 2H, J=7.6Hz), 7.15 (s, 1H), 6.91 (d, 1H, J=8.6 Hz), 6.85 (d, 1H, J=8.6 Hz), 3.32(t, 2H, J=7 Hz), 2.95 (t, 2H, J=7 Hz), 2.18 (s, 3H)); LCMS calc. forC₁₈H₁₆F₃NO₄ (M+H⁺): 368, found 368.

Example 1 Anti-Inflammatory Effect in Cell

1-1. Suppressing Effect on Production of NO

Inhibiting effect of the 2-hydroxybenzoic acid derivative according tothe present invention on NO, inflammatory factor interveninginflammation reaction was evaluated. BV2 microglia cell line was treatedwith lipopolysaccharide (LPS), a inflammation-inducing material ofbacteria toxin, with inclusion of 10 uM, 30 uM or 100 uM of the presentcompound. After 24 hours of incubation, 50 ul of culture medium wascollected in 96-well plate. Then, 50 ul of Griess reagent was added andincubated for 10 minutes at room temperature. The absorbance wasevaluated by ELISA reader at 540 nm. IC₅₀ value of each compound wascalculated and shown in the below table 4 (“NO in BV”).

As shown in the below table 4, IC₅₀ values of compound 9 and 14 were1.79 uM and 6.7 uM, respectively, which are on a highest level. Wheninhibiting effect on the production of NO was evaluated as IC₅₀ value,most compounds had lower values than 100 uM. As shown in results, the2-hydroxybenzoic acid derivative of the present invention is thought tobe useful as anti-inflammatory agent because the compound suppresses theactivity of NO intervening inflammation reaction.

1-2. Suppressing Effect on Gene Expression

Effects of the 2-hydroxybenzoic acid derivatives on iNOS gene expressionin BV2 cell line were evaluated. 100 mm dish was inoculated with 1×10⁶cells of BV2 cell line, and treated with both LPS, inflammation-inducingmaterial, and 1˜110 uM of the 2-hydroxybenzoic acid derivative of thepresent invention, together, in regular concentration interval. After 24hours of incubation, RNA was extracted. Extracted RNA was used inreverse transcription and polymerase chain reaction, and expressionpatterns of TNF-alpha, IL-1beta and iNOS were compared. Results wereshown in the below table 4.

As shown in the table 4, when gene expressions of inflammatory cytokinesare compared to control (LPS treatment), expression of TNFalpha gene wasdecreased 63.24% by compound 2, 77.05% by compound 8, and 61.67% bycompound 9. Expression of IL-1beta gene was evaluated to be decreased75.1% by compound 2, 67.7% by compound 5, and over 50% by compounds 8,9, 10 and 14. Furthermore, compounds 2, 5, 8, 10, 15 and 17 suppressedexpression of iNOS gene by more than 50%.

TABLE 4 Compound IC₅₀ (uM) % Inhibition (in 100 uM) No. NO in BVTNF-alpha IL-1beta iNOS 1 182.99 ND ND ND 2 24.26 63.2 75.1 105.5 326.14 ND ND ND 4 6.25 ND ND ND 5 12.45 38.7 67.7 108.5 6 >100 ND ND ND 723.69 ND ND ND 8 63.25 77.1 56.9 113.8 9 1.79 61.7 76.7  17.8 10 20.67ND 83.6  88.9 11 132.15 ND ND ND 12 18.62 ND 41.8  43.7 13 85.43 43   ND 15.5 14 6.7 38.9 80.4  44.3 15 20.34 37.4 35.1  86.2 16 51.78 ND ND ND17 26.5 20.8 ND 111.1 18 21.63 ND ND ND In the table 4, the term ‘ND’means “not determined.”

In the table 4, the term ‘ND’ means “not determined.”

1-3. Suppressing Effect on Production of Cytokine

Effects of the 2-hydroxybenzoic acid derivatives on production ofcytokine in BV2 cell line were evaluated. 24-well plate was inoculatedwith 1×10⁶ cells of BV2 cell line, and treated with both LPS,inflammation-inducing material, and 100 uM of sample (the2-hydroxybenzoic acid derivative or comparative drug), together. After24 hours of incubation, culture medium was collected. The levels ofcytokines, TNF-α and IL-6, in the medium were evaluated by ELISA method.Results were shown in the below table 5.

As shown in the table 5, when compared to control (LPS treatment), thelevel of TNF-α produced by LPS was reduced 62.26% by compound 2, 31.90%by compound 3, and 62.6% by compound 14. However, comparative drugs(ibuprofen and aspirin) did not showing reducing effect at the sameconcentration. Similarly, the level of IL-6 was apparently decreased bycompound, but the level of IL-6 was little reduced by ibuprofen andaspirin.

TABLE 5 TNF-α IL-6 compound No % Inhibition % Inhibition 2 62.26 70.31 331.90 33.06 4 18.71 ND 14  62.60 ND Ibuprofen No effect No effectAspirin No effect 22.18 In the table 5, the term ‘ND’ means “notdetermined.”

Example 2 Basic Toxicity Test

Single-dose toxicities of the 2-hydroxybenzoic acid derivatives wereevaluated. Results were shown in FIG. 32.

As shown in FIG. 32, LD₅₀ of compounds 2 and 18, preferable examples ofthe present invention, were more than 3 g/kg, which means that thecompounds have good safety. Chemical 19 had 0.5-1 g/kg of LD₅₀, that is,chemical 19 showed relatively worse safety. In addition, compound 4showed much better effect in anti-inflammatory test, etc., but compound4 did not show dose-dependent result in toxicity test because thecompound caused sudden death of mouse at 3 g/kg of dose test. Compounds3 and 14 have the similar chemical structure with compound 2, and showthe similar therapeutic effects with compound 2 in anti-inflammatorytest, but the compounds show high toxicity or dose-independent toxicity.

Example 3 Safety Test About Induction of Gastric Mucous Membrane Damage

Conventional NSAIDs have side effects causing damages to the gastricmucous membrane. Therefore, it was evaluated whether compound 2 havinganti-inflammatory effect causes the gastric damage or not. 30, 100 or300 mg/kg of aspirin was orally administered as control. Compound 2 ofthe present invention did not cause the gastric side effect even when1,000 mg/kg of compound 2 was orally administered. From this result, itis believed that the 2-hydroxybenzoic acid derivative of the presentinvention is very safe (FIG. 1).

In addition, the safety of compound 18 was evaluated by comparison withindomethasin and ibuprofen (other NSAIDs) and celecoxib (a selectiveCOX-2 inhibitor). Compound 18 did not cause damage to gastric mucousmembrane even in the high dose of oral administration (FIG. 2).

Example 4 Cell-Protecting Effect

Cell-protecting effect of the 2-hydroxybenzoic acid derivative of thepresent invention was evaluated. AGS cell line (human gastric cancercells, 1×10⁵ cells) was inoculated onto 96-well plate. Then, death ofgastric mucous membranes was caused by 24 hour-treatment of Helicobacter(FIG. 3), 16 hour-treatment of sulindoc (FIG. 4), 8 hour-treatment ofH₂O₂ (FIG. 5) or 24 hour-treatment of ethanol (FIG. 6). In addition,some of AGS cell line were together treated with 10 uM, 30 uM or 100 uMof compound 2. The cell lines were incubated for 24 hours. MTT solutionwas added to each well, and incubated 4 hours at 37° C. After than, theabsorbance was analyzed by ELISA reader at 540 nm, and the survival rateof gastric mucous membranes cell was calculated. In results, 1 uM and 10uM of compound 2 showed more than 50% of cell-protecting effect, and 100uM of compound 2 showed more than 70% of cell-protecting effect in mostcell death models.

As one marker of inflammation, the expression level of TNF-α mRNA wasevaluated by RT-PCR method. Samples treated with ethanol for 24 hourswere used. 1-100 uM of compound 2 completely suppressed the expressionof TNF-α mRNA (FIG. 7).

From these results, the 2-hydroxybenzoic acid derivative of the presentinvention is thought to be effectively used for suppressing inflammationdisease, particularly, protecting cell and suppressing inflammation ingastritis caused by several reasons. Furthermore, the 2-hydroxybenzoicacid derivative of the present invention showed better effect thanmucosta and PPI, which are known as therapeutic agent for treatinginflammation.

Example 5 Protecting Effect on Gastrointestinal Bleeding

5-1. Gastritis Induced by Alcohol and Hydrochloric Acid

Effect of the 2-hydroxybenzoic acid derivative on gastritis induced byalcohol and hydrochloric acid was evaluated. 200˜250 g of male SD ratwas fasted for 24 hours, and 60% EtOH/150 mM HCl was orally administeredto induce gastric bleeding (gastric injury). Compound 2 was orallyadministered according to indicated dose 30 minutes before the gastricbleeding, and stomach was taken out 90 minutes after gastric bleeding toevaluate the degree of gastric injury. In results, the compound 2protected stomach against gastric bleeding induction, and did not showany toxicity. Results were shown in FIG. 8.

5-2. Alcoholic Gastritis

Effect of the 2-hydroxybenzoic acid derivative on alcoholic gastritiswas evaluated. 250 g of rat was fasted for 24 hours, and alcohol wasorally administered to cause gastric bleeding (B). Some rats werepre-treated with compound 21 hour before the administration of alcohol,and the rat showed significantly reduced gastric bleeding (C).Quantified results were shown in FIG. 9 (D). From these results, thecompound 2 of the present invention is thought to have strong protectingeffect on alcoholic gastritis.

Treatment of alcoholic gastritis is known to be more difficult thangastritis caused by other reasons because it is not easy to be treatedeven with inhibitor of gastric juice secretion or enhancer of gastricprotecting factor. This is because alcohol causes the disease by thetypes of directly mediating toxicity and inflammation, inducingbleeding, etc. The compound 2 of the present invention is thought tosignificantly reduce this type of direct toxicity of alcohol.

5-3. Gastritis Caused by NSAID

Effect of the 2-hydroxybenzoic acid derivative on NSAID-inducedgastritis was evaluated. Compound 2 showed excellent protecting effecton stomach (B and D of FIG. 10) in both weak gastric damage model (A ofFIG. 10) induced by NSAID (indomethacine) 6 hour-administration andsevere gastric damage model (C of FIG. 10) induced by indomethacine 12hour-administration.

Example 6 Efficacy Test in Arthritis Animal Model

6-1. Inflammation Model Induced by Carrageenan: Evaluation of AnalgesicEffect

Analgesic effect of compound 2, one example of the 2-hydroxybenzoic acidderivative, was evaluated in arthritis model. 2% carrageenan wasintradermally injected into left pes of rat, and 3-300 mg/kg of compound2 was orally administered to evaluate effect of compound 2 on thermalhyperalgesia of arthritis model. Results were shown in FIG. 10.

Compound 2 decreased thermal hyperalgesia in a dose-dependent manner.300 mg/kg of compound 2 reduced thermal hyperalgesia by over 90%. Inaddition, the equivalent level of decreasing effect with 50 mg/kg ofibuprofen, a known anti-inflammatory agent, was shown in group having100 mg treatment of compound 2.

6-2. Inflammation Model Induced by Zymosan: Evaluation of Cytokine

Anti-inflammation effect of compound 2 in arthritis model was evaluatedwith zymosan. 0.5 ml of 1% zymosan was administered with air pouch, and3-300 mg/kg of compound 2 was once orally administered 1 hour beforeadministration of zymosan. Edema, a main symptom of arthritis, and thelevel of cytokine were observed 4 hours later. In results, over 30 mg/kgof compound 2 decreased the level of TNF-alpha more than control, anddecreased the level up to 300 mg/kg in a dose-dependent manner. Ascomparative example, ibuprofen (50 mg/kg), a well-knownanti-inflammatory drug, was used (FIG. 12). Similarly, IL-1alpha alsowas decreased in a dose-dependent manner (FIG. 13). From these results,the 2-hydroxybenzoic acid derivative of the present invention is thoughtto be useful for treating inflammatory disease, particularly arthritis.

6-3. Animal Model Evaluation of Arthritis Induced by Collagen

To evaluate the efficacy of the 2-hydroxybenzoic acid derivative,collagen-induced arthritis model (rheumarthritis animal model) was used.Bovine type II collagen was mixed with complete Freund's adjuvant tomake an emulsion, and the emulsion was intradermally injected into theorigin site of 8˜10 week-old DBA/1LacJ mouse tail. Intradermal boostingwas performed by the same method 2 weeks later. 25 mg/kg/day ofcompounds 2, 3 and 18, 1 mg/kg/week of methotrexate (comparativeexample) or 10% vehicle (control) were intraperitoneally injected oneweek after the second intradermal injection of collagen (FIG. 15).Pictures of foot edema of normal rat (sham), collagen-administered rat(collagen) and compound 18-administered rat (compound 18, 25 mg/kg/dayinjection) were shown in FIG. 14.

In addition, 50 mg/kg/day of compounds 6 and 11, and 5 mg/kg/2 days ofmethotrexate (comparative example) were intraperitoneally injected.Phosphate buffered saline was injected as control (FIG. 16). For 2˜3weeks, the degree of arthritis was observed everyday, and the result wasevaluated according to the below arthritic index using edema. As shownin results of arthritic index, compounds 2, 3 and 18, and compounds 6and 11 showed an apparent reducing effect (FIGS. 15 and 16).

—Arthritic Index—

4 paws were evaluated as from 0 to 4 point, Total points: 16

0 point—normal paw

1 point—mild edema and flare limited to tarsal bone

2 points—mild edema and flare extending from ankle joint to tarsal bone

3 points—middle edema and flare extending from ankle joint to metatarsalbone

4 points—edema and flare extending from ankle joint to total digit

Example 7 Efficacy Test in Inflammatory Intestinal Disease Animal Model

5% dextran sulfate sodium (DSS) model was used as inflammatoryintestinal disease animal model. 5% DSS mixed with water was orallyadministered. 5% DSS model causes damage to epithelial cell, and ulcerin left colon and the decrease of colon length are mainly observed inthe model. This model is simple and reproductive, and can regulate thedegree of inflammation according to dose. Therefore, this model is oftenused for evaluating drug candidates.

DSS is continuously administered in water bottle. The 2-hydroxybenzoicacid derivative was suspended in 10% vehicle, and orally administered.

Administration of DSS changed the length and width of colon. Treatmentof compound 18 significantly changed the length and width of the colon(FIGS. 17, 18 and 19). This result means that the compound of thepresent invention has protective effect in this colitis model.

In addition, bloody excrement, diarrhea and the degree of dirtiness wereevaluated everyday as feces condition. Both sulfasalazine and compound18 significantly decreased feces score compared to group treated withDSS only (FIG. 20).

Example 8 Efficacy Test in Acute Pancreatitis Animal Model

Cerulein-induced pancreatitis model was used as acute pancreatitismodel. Cerulein is an analog of cholecystokinin and a hormone causingsecretion of pancreatic digestive enzyme in pancreas. Digestive enzymewas over-secreted into pancreas by intraperitoneal injection of cerulein(50 ug/kg). LPS was injected 2 hours later to induce edema. Whencerulein was administered, massive edema was observed on the basis ofspleen. In addition, when 25 mg/kg of compound 18 was administered, theedema was decreased (FIG. 21). D of FIG. 21 is results showing thepancreas weight increased by the edema. Group treated with compound 18had significantly reduced pancreas weight.

FIG. 22 shows the levels of amylase and lipase, pancreatic digestiveenzymes, in plasma. The model excessively increases digestive enzymes,which are released into blood. That is, the decrease of digestive enzymein blood can be an indirect evidence for sample to reduce pancreatitis.As shown in FIG. 22, amylase and lipase were increased in the blood ofthe pancreatitis animal model, and the administration of 25 mg/kg ofcompound 18 significantly reduced the increase.

To confirm the protecting effect on inflammation, the levels of TNF-αand IL-1β, inflammatory cytokines, were evaluated. PGE₂, the product ofCOX-2 (inflammation enzyme), were evaluated by ELISA method. TNF-α,IL-1β and PGE₂ were excessively increased in the pancreatitis model, andcompound 18 significantly reduced these inflammation markers (FIGS. 23and 24).

Example 9 Efficacy Test of Compound 2 in APP_(swe)/PS1_(deltE9) DoubleTransgenic Dementia Mice

9-1. Reduction Evaluation of Cytokines by ELISA Method

APP/PS1 transgenic dementia mice were fed chow containing 25 mg/kg/dayof compound 2 or 62.5 mg/kg/day of ibuprofen, for 7 months before beingsacrificed (3.5˜10.5 months). After administration of drug for 7 months,the levels of TNF-α, IL-1β and IL-6 were quantified by ELISA (mean±SEM,n=3-5). Significant difference from control (APP/PS1 mouse fed withgeneral chow only), p<0.05 using one-way ANOVA according toStudent-Neuman-Keuls' test.

In result, treatment with compound 2 significantly reduced the levels ofTNF-α, IL-1β and IL-6 compared to APP/PS1 mouse fed with general chowonly (FIG. 25).

9-2. Protecting Effect of Compound 2 on Brain-Blood Vessel Barrier

Damage of brain blood vessel is well known in Alzheimer's disease.Protecting effects of compound 2 and ibuprofen (comparative example) ondamage of brain-blood vessel barrier were evaluated. 3 ml/g of 2% Evansblue dye was administered into blood vessel of 5 month-old APP/PS1dementia mouse. Chow containing 25 mg/kg/day of compound 2 or 62.5mg/kg/day of ibuprofen was provided from 2 month-old to 5 month-old. Inresults, as shown in B and F of FIG. 26, the permeability of Evans bluedye was increased in cortex, hippocampus and thalamus, compared tonormal mouse. In addition, as shown in FIG. 26, protecting effects onbrain-blood vessel barrier were shown in group treated with compound 2(C and G of FIG. 26) and group treated with ibuprofen (D and H of FIG.26).

9-3. Reduction Evaluation of Compound 2 on Amyloid Plaque byThioflavin-S Stain Analysis

Effect of the 2-hydroxybenzoic acid derivative on dementia was evaluatedwith Thioflavin-S stain analysis. The treatment with 25 mg/kg/day ofcompound 2 for 7 months (from 3.5 to 10.5 month-old APP/PS1) caused asignificant 53% reduction in amyloid plaque burden compared to APP/PS1dementia mouse fed with general chow only (FIG. 27). In addition, thetreatment with 25 mg/kg/day of compound 2 for 4 months (from 8.5 to 12.5month-old APP/PS1) caused a significant 49.3% reduction in amyloidplaque burden compared to APP/PS1 dementia mouse fed with general chowonly.

9-4. Behavior Improvement of Compound 2 by Elevated Plus Maze Test

APP/PS1 transgenic mice were fed chow alone or containing 25 mg/kg/dayof compound 2, for 7 months (3.5-10.5 months). After administration of 7months, Elevated plus maze test was performed to evaluate the behaviorimprovement. Elevated plus maze has two open arms (30 cm×6 cm×0.5 cm)and two closed arms (30 cm×6 cm×15 cm), and also has 6 cm×6 cm of centerplatform. In Elevated plus maze test, mouse was carefully laid in thecenter with the head of the mouse toward open arm. The time that themouse spent in the open arm was recorded for 5 minutes (mean±SEM,n=3-5). *, Significant difference from control (APP/PS1 mouse fed withgeneral chow only), p<0.05 using one-way ANOVA according toStudent-Neuman-Keuls' test.

In result, the group treated with 25 mg/kg/day of compound 2 for 7months decreased the time for mouse to stay in the open arm compared tothe group provided with chow only (FIG. 28).

Example 10 Efficacy Test of Compound 2 in G93A ALS Animal Model

10-1. Reduction of Microglia Activation

G93A (Glycine

Alanine) transgenic mouse having similar pathophysiologicalcharacteristics with ALS (amyotrophic lateral sclerosis) human patientwas used to evaluate therapeutic effect of drug in ALS, one of maindegenerative brain diseases.

Activation degree of microglia (a marker of inflammation in braindisease model) expressed in the lumbar ventral horn of G93A mouse wereevaluated with TOMATO Lectin dye. In G93A mouse, microglia was moreactivated compared to the wild type mouse. In addition, treatment with 5mg/kg/day of compound 2 decreased the number and activation degree ofmicroglia (FIG. 29).

10-2. Reduction of Cytokine Expression

Lumbar segments of 16 week-old G93A mice fed with general chow only, and16 week-old G93A mice fed with chow containing 5 mg/kg/day of compound 2were extracted and their RNA were separated. The mRNA expression degreesof TNF-α and IL-1β, inflammatory cytokines, were evaluated throughRT-PCT. In results, administration of compound 2 effectively reducedinflammatory cytokines (FIG. 30).

Example 12 Efficacy Test of Compound 2 in Parkinson's Disease AnimalModel

MPTP (40 mg/kg) was subcutaneously injected into C57/BL6 mice (male/8week-old). Some of the mice were administered with 50 mg/kg of compound2 through intraperitoneal injection 30 minutes before the injection ofMPTP everyday. Two days later, brain tissue was extracted andimmunostained with CD11b. After reaction, DAB (diaminobenzidine) wasused for chromograph, and then the activation degree of microglia (amarker of inflammation in brain disease model) was evaluated withoptical microscope (FIG. 31).

In result, the activity of microglia caused by MPTP was decreased by theadministration of compound 2.

The concrete diseases applicable with the compound or itspharmaceutically acceptable salt of the present invention are describedas follows. However, the scope of the present invention is not limitedto the diseases described below.

Application Example 1 Gastritis

The 2-hydroxybenzoic acid derivative of the present inventioneffectively suppressed cell death in experiments using cultured cell,induced by alcohol, Helicobacter Pylori, NSAID and H₂O₂. The compound ofthe present invention showed better effect than mucosta or PPI, knowndrugs having cell-protecting effect on gastritis. In addition, thecompound of the present invention showed superior therapeutic effect onalcohol-induced gastritis in animal model, and alleviated gastritisinduced by NSAID. The compound of the present invention also showed goodsafety in comparative experiment using aspirin because the compound didnot any gastric bleeding even at much higher dose than that of aspirin.Therefore, the 2-hydroxybenzoic acid derivative of the present inventionis thought to be useful for treating or preventing gastritis.

Application Example 2 Inflammatory Bowl Disease (IBD)

Prostaglandin (the product of cyclooxygenase (COX)) and leucotriene (theproduct of lipooxygenase) are known to take an important part ininflammation of inflammatory diseases like ulcerative colon. Zileuton,an inhibitor of 5-lipooxygenase (5-LOX), and sulfasalazine decreased theactivity of MPO (myeloperoxidase) which is used as a marker showing thedegree of inflammation in intestinal damage animal model (Singh V P etal., Indian J Exp Biol. 2004; 42(7):667-73). In addition, nimesulide (aselective COX-2 inhibitor) showed protective effect in two intestinaldamage animal models (acetic acid-induced IBD and LTB4-induced IBD).Nimesulide excessively suppressed the activity of MPO in inflammationreaction (Singh V P et al., Prostaglandins Other Lipid Mediat. 2003;71(3-4):163-75). Therefore, the compound of the present invention havinganti-inflammatory effect can be effectively used for treatinginflammatory bowl disease.

Application Example 3 Rheumarthritis

The 2-hydroxybenzoic acid derivative of the present invention showedsimilar or superior therapeutic effect in collagen-induced arthritismodel compared to methotrexate (control), which is used for treatingarthritis, but has adverse effects. In addition, the compound of thepresent invention alleviated pain of hyperalgesia induced by carrageenanand suppressed the production of inflammatory cytokines induced byzymosan. Therefore, the compound of the present invention having thesimilar efficacy with known anti-inflammatory agents and good safety canbe used as therapeutic agent for arthritis.

Application Example 4 Pancreatitis

Acute pancreatitis is an inflammation related with pancreasautodigestion caused by reflux of digestive enzyme of pancreatic juiceor bile of cholelithiasis into pancreas. Pancreatitis shows varioussymptoms like from mile edema to severe bleeding, which cause severaldamages to pancreas. There are a lot of evidences showing thatpancreatitis is related with inflammation, and it is reported that COXinhibitor has a protective effect in pancreatitis model and suppressesthe production of inflammatory markers, TNF-α and prostaglandin (Song AM et al., Am J Physiol Gastrointest Liver Physiol. 2002;283(5):G1166-74). Therefore, the compound of the present invention canbe effectively used for treating pancreatitis.

Application Example 5 Diabetic Inflammation and Pain

Roles of inflammation and pain are becoming important in type IIdiabetes. There are lots of literatures reporting that many drugs havinganti-inflammatory effect reduce sign of type II diabetes or delay onsetof type II diabetes (Deans K A et al., Diabetes Technol Ther. 2006;8(1):18-27). Lisofylline, an anti-inflammatory compound, reduceddiabetic symptoms 50% in mouse administered streptozotocin bysuppressing IFN-gamma and TNF-alpha (Yang Z et al., Pancreas. 2003;26(4):e99-104). Therefore, the compound of the present invention havinganti-inflammatory effect can be effectively used for treating orpreventing diabetic inflammation and pain.

Application Example 6 Arteriosclerosis

In early formation of atherosclerotic lesion of apolipoproteinE-deficient (apoE(−/−)) mouse, selective COX-2 inhibitor (for example,rofecoxib and NS-398) and non-selective COX inhibitor (for example,indomethacine) reduced atherosclerosis by about 35-38% and about 38-51%,respectively (Burleigh M E J Mol Cell Cardiol. 2005 September;39(3):443-52). Therefore, the compound of the present invention havinganti-inflammatory effect can be effectively used for treatingarteriosclerosis.

Application Example 7 Alzheimer's Disease

Alzheimer's disease is the most common form of adult onset dementia.Alzheimer's disease is characterized as the presence of theneurofibrillary tangles, amyloid plaques and severe neuronal death.

Also, there are lots of literatures showing that Alzheimer's disease isrelated with inflammation. It were often reported that microglia andinflammatory cytokines were increased in dementia animal model(Minghetti L. Current Opinion in Neurology 2005, 18:315-321), and thatan administration of drug inhibiting inflammation may have a protectingeffect in animal models of Alzheimer's disease (Townsend K P and PraticoD. FASEB J. 2005; 19(12): 1592-601).

Therefore, the compound of the present invention having cell-protectingeffect and anti-inflammatory effect can be effectively used for treatingAlzheimer's disease.

Application Example 8 ALS

Lou Gehrig Disease is named amyotrophic lateral sclerosis or motorneuron disease, and the progressive degeneration of motorneurones is thepathological hallmark of this disease.

There are lots of literatures showing that ALS is related withinflammation. It were often reported that microglia and inflammatorycytokines were increased in G93A mouse, animal model of ALS (Weydt P andMoller T. Neuroreport. 2005, 25; 16(6): 527-31), and an administrationof drug inhibiting inflammation may have a protecting effect in animalmodels of ALS (West M et al., J Neurochem. 2004; 91(1): 133-43).

Therefore, the compound of the present invention can be effectively usedfor treating ALS.

Application Example 9 Parkinson's Disease

Parkinson's Disease (PD), the prototypic movement disorder, ischaracterized clinically by tremor, rigidity, bradykinesia and posturalinstability and diagnosed pathologically by a selective death ofdopaminergic neurons in the substantia nigra.

There are lots of literatures showing that Parkinson's disease isrelated with inflammation. It were often reported that microglia andinflammatory cytokines were increased in Parkinson's disease (Gao H M,Trends Pharmacol Sci. 2003; 24(8): 395-401; Minghetti L. Curr OpinNeurol. 2005; 18(3):315-21), and an administration of drug inhibitinginflammation may have a protecting effect in animal models ofParkinson's disease (Gao H M, Trends Pharmacol Sci. 2003; 24(8):395-401).

Therefore, the compound of the present invention having cell-protectingeffect and anti-inflammatory effect can be effectively used for treatingParkinson's disease.

INDUSTRIAL APPLICABILITY

The present invention provide a pharmaceutical composition useful fortreating or preventing inflammatory disease, comprising the2-hydroxybenzoic acid derivative represented by the chemical formula 1or its pharmaceutically acceptable salt, and a method for treating orpreventing inflammatory disease, using the pharmaceutical composition.The pharmaceutical composition of the present invention is very usefulfor treating or preventing inflammatory disease such as gastritis,pancreatitis, colitis, arthritis, diabetic inflammation,arteriosclerosis, nephritis, hepatitis, Alzheimer's disease, Parkinson'sdisease and Lou Gehrig's disease, as well as safe.

1. 2-Acetoxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acidand its pharmaceutically acceptable salts.
 2. A method for manufacturing2-acetoxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid,comprising (S1) reacting2-hydroxy-5-[2-(4-trifluoromethyl-phenyl)-ethylamino]-benzoic acid withdi-tert-butyl dicarbonate; (S2) reacting5-{tert-butoxycarbonyl[2-(4-trifluoromethyl-phenyl)-ethyl]amino}-2-hydroxybenzoicacid, which is made in the (S1) step, with acetyl halide; and (S3)removing tert-butoxycarbonyl group from5-{tert-butoxycarbonyl[2-(4-trifluoromethyl-phenyl)-ethyl]amino}-2-acetoxybenzoicacid, which is made in the (S2) step.